CW-EPR spectra of N6-spin-labeled coenzyme NAD+ (SL-NAD+) bound to microcrystalline, tetrameric glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been collected at 9.8, 34, and 94 GHz. These data have been analyzed, using a combination of the methods of simulated annealing and global analysis, employing a model with pairwise R-axis related dipolar interactions between the nitroxides of N6-SL-NAD+ bound to the enzyme tetramer. Using this approach, a unique fit to the data has been obtained which is consistent with a molecular model built from the known crystal structure of the DAPDH tetramer.